Polo kinases are an evolutionarily conserved subfamily of Ser/Thr protein kinases that play pivotal roles in cellular proliferation. In addition to a high degree of sequence similarity in the kinase domain, they contain a highly conserved sequence motif termed "polo-box" in the non-catalytic C-terminal domain. The polo-box appears to be critical for targeting the catalytic activity of polo kinases to centrosomes, kinetochores, and midbody. However, how polo-box regulates the subcellular localization and which proteins it interacts with are not clearly understood. Here we report the isolation of hCenexin as a novel polo-box interacting protein by a yeast two-hybrid screening. hCenexin localized only to the mother centriole in S and early G2, and to both centrosomes after maturation in G2/M. Both in vivo co-immunoprecipitation and in vitro binding studies showed that Plk1 specifically interacted with the C-terminal domain of hCenexin. Down-regulation of hCenexin by hCenexin RNAi resulted in delocalization of hPlk1 from the centrosomes, but not from kinetochores and midbodies. Consistent with the role of Plk1 in establishing spindle bipolarity, hCenexin RNAi cells exhibited delayed microtubule nucleation. These data suggest that hCenexin interacts with the polo-box domain of Plk1, and this interaction is required for proper subcellular localization and mitotic functions of Plk1. Whether this localization is critical for specific functions of Plk1-mediated mitotic events is currently under investigation.